Use of recombinant collagen in preparation of preparation for increasing expression of skin fibroblast proliferation related genes

Abstract

The present application provides the use of a recombinant collagen in the preparation of a preparation for increasing the expression of skin fibroblast proliferation-related genes, wherein the amino acid sequence of the recombinant collagen comprises SEQ ID NO: 1. The present application detects the expression of a series of genes capable of enhancing the proliferation of skin fibroblasts by transcriptomics, and proves that the recombinant collagen can promote the expression of skin fibroblast proliferation genes. The collagen can be used for the development and application of various preparation products, and endows the preparation products with the efficacy and effect of promoting the proliferation of skin fibroblasts.

Description

Technical Field

[0001] The present invention belongs to the field of research on the efficacy of cosmetic raw materials, and specifically relates to the use of recombinant collagen in the preparation of a preparation for increasing the expression of genes related to the proliferation of skin fibroblasts. Background Art

[0002] Collagen is a family of proteins. At least 30 types of collagen chain coding genes have been discovered, which can form more than 16 types of collagen molecules. According to their structure, they can be divided into fibrous collagen, basement membrane collagen, microfibrillar collagen, anchoring collagen, hexagonal reticular collagen, non-fibrous collagen, transmembrane collagen, etc.

[0003] Based on their distribution and functional characteristics within the body, collagen can be divided into interstitial collagen, basement membrane collagen, and pericellular collagen. Interstitial collagen molecules comprise the vast majority of collagen in the body and include types I, II, and III. Collagen's excellent biocompatibility, biodegradability, and bioactivity have led to its widespread application in food, medicine, tissue engineering, cosmetics, and other fields.

[0004] Fibroblasts (found in the dermis) are the main cellular components of loose connective tissue. Fibroblasts synthesize and secrete collagen and elastin, generating collagen fibers, reticular fibers, and elastic fibers. They also synthesize and secrete matrix components such as glycosaminoglycans and glycoproteins. Collagen peptides have biological activities such as repairing skin damage, antioxidant activity, antihypertensive activity, and fat-reducing activity.

[0005] Currently, collagen is primarily produced and isolated from tissues of animals such as fish, pigs, and cattle, which can pose a risk of viral transmission. Furthermore, due to the diverse sources of animal collagen, quality control is difficult and the production process is complex. Therefore, providing a recombinant collagen produced using genetic engineering techniques has significant application value. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide the use of recombinant collagen in the preparation of a preparation that increases the expression of genes related to skin fibroblast proliferation. The present invention uses transcriptomics to detect the expression of a series of genes that can enhance skin fibroblast proliferation, demonstrating that recombinant collagen SEQ ID NO: 1 can promote the expression of skin fibroblast proliferation genes. This collagen can be used in the development and application of a variety of cosmetic products, giving cosmetic products the efficacy and effect of promoting skin fibroblast proliferation. At the same time, this series of genes can also serve as a reference for future evaluation of the ability of other raw materials or cosmetics to enhance skin fibroblast proliferation.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a use of recombinant collagen in preparing a preparation for increasing the expression of genes related to skin fibroblast proliferation, wherein the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO: 1.

[0009] Preferably, the skin fibroblast proliferation-related genes include: any one or a combination of at least two of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA.

[0010] The present invention uses transcriptomics to examine the expression of a series of genes that enhance skin fibroblast proliferation. The results show that the recombinant collagen protein can promote the expression of skin fibroblast proliferation genes. This collagen protein can be used in the development and application of various cosmetic products, endowing these cosmetic products with the efficacy and effect of promoting skin fibroblast proliferation.

[0011] Preferably, the skin fibroblast proliferation-related genes include: a combination of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 and VEGFA.

[0012] Preferably, the formulation comprises a pharmaceutical or cosmetic product.

[0013] Preferably, the concentration of the recombinant collagen in the preparation is 0.4-0.6 mg / mL, for example, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL or 0.6 mg / mL.

[0014] Preferably, the cosmetics include: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

[0015] The present invention also provides an expression promoter for increasing the expression of genes related to skin fibroblast proliferation. The expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO: 1.

[0016] In the present invention, after the recombinant collagen is administered, the relative expression level of the skin fibroblast proliferation-related gene is 1.821.

[0017] In the present invention, a relative expression promotion of 1.19 is considered to be promotion. In bioinformatics analysis, a log2 foldchange value of 0.25 is usually used as a threshold for screening differentially expressed genes, which is converted into a relative expression fold of about 1.19.

[0018] The present invention provides a biomarker for evaluating the ability of cosmetics or their raw materials to enhance the proliferation of skin fibroblasts. The biomarker is a skin fibroblast proliferation-related gene, and the gene includes: any one of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA, or a combination of at least two of them.

[0019] In a second aspect, the present invention provides the use of a product for detecting the expression level of a biomarker in evaluating the ability of cosmetics or their raw materials to enhance the proliferation of skin fibroblasts, wherein the biomarker is a gene related to the proliferation of skin fibroblasts, and the gene includes: any one or a combination of at least two of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA.

[0020] Preferably, the genes include: a combination of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 and VEGFA.

[0021] Preferably, the product comprises a primer set, a reagent or a detection model.

[0022] Preferably, the reagents include qPCR detection reagents.

[0023] In a third aspect, the present application provides a primer combination for detecting the expression level of a biomarker, the primer combination comprising:

[0024] an upstream primer for amplifying BMP2 as set forth in SEQ ID NO: 5 and a downstream primer as set forth in SEQ ID NO: 6;

[0025] an upstream primer for amplifying CCL2 as set forth in SEQ ID NO: 7 and a downstream primer as set forth in SEQ ID NO: 8;

[0026] an upstream primer for amplifying CCN1 as set forth in SEQ ID NO: 9 and a downstream primer as set forth in SEQ ID NO: 10;

[0027] an upstream primer for amplifying CCND1 as set forth in SEQ ID NO: 11 and a downstream primer as set forth in SEQ ID NO: 12;

[0028] an upstream primer for amplifying EDN1 as set forth in SEQ ID NO: 13 and a downstream primer as set forth in SEQ ID NO: 14;

[0029] an upstream primer for amplifying EDN2 as set forth in SEQ ID NO: 15 and a downstream primer as set forth in SEQ ID NO: 16;

[0030] an upstream primer for amplifying FGF1 as set forth in SEQ ID NO: 17 and a downstream primer as set forth in SEQ ID NO: 18;

[0031] an upstream primer for amplifying FOXC2 as set forth in SEQ ID NO: 19 and a downstream primer as set forth in SEQ ID NO: 20;

[0032] an upstream primer for amplifying FOXF1 as set forth in SEQ ID NO: 21 and a downstream primer as set forth in SEQ ID NO: 22;

[0033] an upstream primer for amplifying HGF as set forth in SEQ ID NO: 23 and a downstream primer as set forth in SEQ ID NO: 24;

[0034] an upstream primer for amplifying KLF4 as set forth in SEQ ID NO: 25 and a downstream primer as set forth in SEQ ID NO: 26;

[0035] an upstream primer for amplifying LIF as set forth in SEQ ID NO: 27 and a downstream primer as set forth in SEQ ID NO: 28;

[0036] an upstream primer for amplifying MYC as set forth in SEQ ID NO: 29 and a downstream primer as set forth in SEQ ID NO: 30;

[0037] The upstream primer for amplifying NGF is shown in SEQ ID NO:31, and the downstream primer is shown in SEQ ID NO:32;

[0038] The upstream primer for amplifying NOG is shown in SEQ ID NO:33, and the downstream primer is shown in SEQ ID NO:34;

[0039] The upstream primer for amplifying PDGFA is shown in SEQ ID NO:35, and the downstream primer is shown in SEQ ID NO:36; the upstream primer for amplifying PDGFB is shown in SEQ ID NO:37, and the downstream primer is shown in SEQ ID NO:38; the upstream primer for amplifying RUNX2 is shown in SEQ ID NO:39, and the downstream primer is shown in SEQ ID NO:40; the upstream primer for amplifying SERPINE1 is shown in SEQ ID NO:41, and the downstream primer is shown in SEQ ID NO:42; the upstream primer for amplifying SFRP1 is shown in SEQ ID NO:43, and the downstream primer is shown in SEQ ID NO:44;

[0040] The upstream primer for amplifying SMAD3 is shown in SEQ ID NO:45, and the downstream primer is shown in SEQ ID NO:46; the upstream primer for amplifying SNAI1 is shown in SEQ ID NO:47, and the downstream primer is shown in SEQ ID NO:48;

[0041] The upstream primer for amplifying STAT1 is shown in SEQ ID NO:49, and the downstream primer is shown in SEQ ID NO:50;

[0042] The upstream primer for amplifying STAT3 is shown in SEQ ID NO:51, and the downstream primer is shown in SEQ ID NO:52;

[0043] The upstream primer for amplifying TGFB1 is shown in SEQ ID NO:53, and the downstream primer is shown in SEQ ID NO:54;

[0044] The upstream primer for amplifying TGFB2 is shown in SEQ ID NO:55, and the downstream primer is shown in SEQ ID NO:56;

[0045] The upstream primer for amplifying VEGFA is shown in SEQ ID NO: 57, and the downstream primer is shown in SEQ ID NO: 58. The primers are shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049] The present invention can quickly use qPCR experiments to verify the ability of new raw materials or products to promote fibroblast proliferation by screening primers for a series of genes, and ensure the accuracy of experimental results.

[0050] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention screens a group of genes that promote fibroblast proliferation through transcriptomics, which can be used as detection targets for promoting fibroblast proliferation in other cosmetic raw materials or finished products in the future.

[0053] The present invention develops a new collagen that exhibits a better function in promoting fibroblast proliferation than existing collagen, and can be added to skin care products as a raw material in the future to achieve better skin care effects.

[0054] The present invention designs and summarizes a series of primers for genes that promote fibroblast proliferation, providing convenience for rapid and accurate detection of related genes in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Heat maps of the experimental and control groups.

[0056] Figure 2 The figure shows the fibroblast proliferation experiment results of the control group at 0 hours.

[0057] Figure 3 The figure shows the results of 24-hour fibroblast proliferation experiment in the control group.

[0058] Figure 4 These are the results of the fibroblast proliferation experiment in the experimental group at 0.5 mg / mL and 0 hours.

[0059] Figure 5 These are the results of the fibroblast proliferation experiment in the experimental group at 0.5 mg / mL and 24 hours.

[0060] Figure 6 These are the results of the fibroblast proliferation experiment in the experimental group at 0.2 mg / mL and 0 hours.

[0061] Figure 7 These are the results of the fibroblast proliferation experiment in the experimental group at 0.2 mg / mL and 24 hours.

[0062] Figure 8These are the results of the fibroblast proliferation experiment in the experimental group at 0.8 mg / mL and 0 hours.

[0063] Figure 9 These are the results of the fibroblast proliferation experiment in the experimental group at 0.8 mg / mL and 24 hours. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0065] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0066] Example 1

[0067] In this example, an expression promoter for increasing the expression of genes related to skin fibroblast proliferation is prepared. The expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen is shown in SEQ ID NO: 1.

[0068] 1. Fermentation process

[0069] First, strain activation was performed, including streaking onto YPD plates and incubating at 30°C for 72 hours. Seed culture preparation was then performed, including selecting a single, well-growing colony in a laminar flow hood and inoculating it into a flask containing YPD medium. The culture was shaken at 30°C, 250 rpm, for 30 hours. The fermentor was then inoculated, and the seed culture was transferred to a fermentor containing 40 g / L glycerol and 4.35 mL / L BSM. Fermentation process control is shown in Table 2.

[0070] Table 2

[0071]

[0072] The experimental samples and equipment include: a ceramic membrane with a pore size of 0.45 μm, a Tofflon hydrophobic chromatography column Phevl Purose6FF; and PrePack50 / 200.

[0073] 2. Purification process

[0074] (1) The fermentation broth was first filtered using a 0.45 μm ceramic membrane and then subjected to hydrophobic chromatography. Sample and column specifications are shown in Table 3.

[0075] Table 3

[0076] Sample Recombinant collagen fermentation broth Filler Phenyl Purose 6FF Column specifications PrePack 50x200 Loading amount 2 CV Experimental flow rate 20 mL / min Detection wavelength 220 nm

[0077] (2) Preparation of experimental materials and buffer solution:

[0078] Distilled water: 5L;

[0079] Equilibration buffer: 20 mM PB, 1 M ammonium sulfate, pH 7, 5 L;

[0080] Elution buffer: 20 mM PB, pH 7, 5 L;

[0081] Wash buffer: 1 M NaOH, 1 L;

[0082] Note: The above buffers must be filtered through a 0.45 μm filter membrane.

[0083] (3) The purification experimental steps are as follows:

[0084] 1) Flush the column with equilibration buffer at a linear flow rate of 90 cm / h for 3 CV (column volume) until the pH, UV, and Cond (conductivity) detection baselines are stable and consistent with the equilibration buffer.

[0085] 2) Load the sample and equilibration buffer at a ratio of 1:1 at a rate of 60 cm / h (20 mL / min), collect the flow-through fractions during the loading process, and detect the purity and content of the target protein.

[0086] 3) After loading, flush with equilibration buffer at a rate of 90 cm / h (30 mL / min) for 2 CV to wash out unbound materials until the UV detection value drops to the initial value.

[0087] 4) Elute the target protein at a rate of 90 cm / h (30 mL / min) with the elution buffer and equilibration buffer in a ratio of 6:4, and collect the eluted fractions.

[0088] 5) Wash the column with at least 2 column volumes of 1 M NaOH at a rate of 60 cm / h (20 mL / min) to remove residual contaminants in the packed column, such as lipids, endotoxins, and nucleic acids.

[0089] 6) Wash the column with 5-10 CV of distilled water at a rate of 60 cm / h (20 mL / min) until the effluent pH is neutral and the Cond (conductivity) is close to 0 to remove 1 M NaOH.

[0090] 7) Wash with 3CV of 20% ethanol to prevent microbial growth.

[0091] Example 2

[0092] This example uses transcriptome sequencing to study the effect of recombinant collagen on the proliferation of skin fibroblasts in Example 1.

[0093] The recombinant collagen was prepared into a solution with a concentration of 0.5 mg / mL, and was added to the cell culture medium of BJ cells (human skin fibroblasts) growing on the substrate to about 70% of the bottom area of the culture dish at a final concentration of 0.1%, and after 24 hours of culture, lysis was performed with Trizon, and the cell lysate was sent to Huada Gene for transcriptome sequencing. The analysis scheme is shown below.

[0094] 1. Analysis scheme

[0095] 1.1 Species name: Homo sapiens; source: NCBI; reference genome version: GCF_000001405.40_GRCh38.p14.

[0096] 1.2 Differential group setting

[0097] One of the important analysis points of transcriptome sequencing is the differential comparison of expression amount. The control and treatment settings in pairwise comparison are shown in Table 4.

[0098] Table 4

[0099] Number Control Treatment 1 BJ_untreated (BJ cells untreated) BJ_treated (BJ cells treated)

[0100] 2. Experimental procedure

[0101] 2.1 mRNA library construction flow

[0102] 1) Total RNA is treated with mRNA enrichment method or rRNA removal method; mRNA enrichment: mRNA with polyA tail is enriched with OligodT magnetic beads; rRNA removal: rRNA is hybridized with DNA probe, DNA / RNA hybrid chain is selectively digested by RNaseH, and DNA probe is digested by DNaseI, and the desired RNA is obtained after purification.

[0103] 2) The obtained RNA is fragmented with fragmentation buffer, and random N6 primers are used for reverse transcription, and then cDNA double strands are synthesized to form double-stranded DNA.

[0104] 3) The synthesized double-stranded DNA is end-repaired and 5'-phosphorylated, and a 3' end is formed with a "A" overhanging sticky end, and then a 3' end with a "T" overhanging bubble-shaped linker is connected.

[0105] 4) The ligation product is amplified by PCR with specific primers.

[0106] 5) The PCR product is heat denatured into single-stranded DNA, and then a bridge primer is used to circularize the single-stranded DNA to obtain a single-stranded circular DNA library.

[0107] 6) Sequencing on machine.

[0108] 4) Data analysis and results

[0109] 4.1 Data analysis process

[0110] The raw data obtained by sequencing is called raw reads. First, we filter out low-quality, adapter contamination, and too high content of unknown bases N reads, and the filtered data is called clean reads, then align the clean reads to the reference genome, and then perform new transcript prediction, SNP & InDel and differential splicing gene detection. After obtaining the new transcripts, we add the new transcripts with protein coding potential to the reference gene sequence to form a complete reference sequence, and then calculate the gene expression level. Finally, for multiple samples, according to the needs, detect the differentially expressed genes between different samples, and do in-depth cluster analysis and functional enrichment analysis of the differentially expressed genes, etc.

[0111] 4.2 The transcriptomic sequencing results obtained are retained for data with p value > 0.05. The FPKM values and log2 fold change values of skin fibroblast proliferation related genes in the experimental group and the control group are extracted.

[0112] 4.3 The FPKM values of the above genes in the experimental group and the control group are integrated into the same table, see Table 5. Through the heat map analysis of the Genedenovo Bioinformatics Cloud tool, a heat map is obtained, as shown in Figure 1 .

[0113] Table 5

[0114]

[0115]

[0116] From Table 5, it can be seen that the final calculation of log2 fold change is increased by about 0.777, that is, the gene expression is promoted by about 1.713 times.

[0117] In this embodiment, the expression of a series of skin fibroblast proliferation related genes is detected by transcriptomics, which proves that the recombinant collagen can enhance the proliferation ability of skin fibroblasts.

[0118] Example 3

[0119] In this embodiment, the recombinant collagen obtained in Example 1 is used for experiments to verify the promotion effect of different concentrations of recombinant collagen on the expression amount of a series of skin fibroblast proliferation related genes. The experimental steps include:

[0120] 1. Cell sample culture

[0121] Collagen was prepared into test solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, respectively. The test solutions were added to the culture medium of BJ cells that were growing adherently to approximately 70% of the bottom area of ​​the culture dish at a final concentration of 0.1% and cultured for 24 hours.

[0122] 2. Extraction and purification of total intracellular RNA

[0123] (1) Remove the culture medium from the cultured cells, wash twice with pre-cooled PBS, add 1 mL of TRIzol reagent, pipette and collect into a centrifuge tube, and place at room temperature for 5 minutes.

[0124] (2) Add 200 μL of chloroform to the centrifuge tube, shake the tube vigorously for 15 seconds, let it stand for 10 minutes, and then centrifuge it at 12,000 g for 15 minutes at 4°C.

[0125] (3) After centrifugation, the liquid is separated into layers. RNA is present in the upper colorless and transparent aqueous layer with a volume of about 600 μL. After transferring to a new centrifuge tube, 500 μL of isopropanol is added and the tube is slowly inverted several times to mix. After standing for 15 minutes, centrifuge at 12,000 g for 10 minutes at 4°C.

[0126] (4) After centrifugation, the RNA precipitates at the bottom of the centrifuge tube, which can be seen as a gelatinous precipitate facing the light source. After aspirating the supernatant, add 1 mL of 75% ethanol to resuspend and wash the RNA, and centrifuge at 7500g for 5 minutes at 4°C.

[0127] (5) Remove the ethanol supernatant, let it stand for 5 minutes to dry the RNA, and add 20 μL of DEPC-water to dissolve the RNA.

[0128] 3. RNA Concentration Determination

[0129] (1) Take 1 μL of the extracted RNA and add it to 99 μL of DEPC-water to make a total volume of 100 μL. Add the diluted RNA solution to a 96-well UV plate.

[0130] (2) Place the 96-well plate in a full-wavelength microplate reader, select the area to be measured, and set the program as follows: oscillate for 10 seconds, measure the absorbance at 260 nm, measure the absorbance at 280 nm, and read the value.

[0131] (3) Calculate OD 260 / OD 280 A ratio between 1.8 and 2.0 indicates that the quality of the extracted RNA meets subsequent needs.

[0132] (4) According to the formula OD260 × 0.04 × dilution factor to calculate the RNA solution concentration in μg / μL. Dilute the RNA with DEPC-water to adjust the concentration to 1 μg / μL.

[0133] 4. Reverse transcription

[0134] (1) Using a reverse transcription kit (Novozymes' one-step reverse transcription kit R333), prepare a reverse transcription system in a 200 μL PCR tube, containing: a) 1 μg / μL RNA solution, 1 μL; b) 5×qRT SuperMix, 4 μL; c) premixed enzyme solution, 1 μL; d) DEPC-water, 14 μL. The final volume of the reverse transcription system is 20 μL. Gently pipette to mix thoroughly and briefly centrifuge for 30 seconds.

[0135] (2) Place the reverse transcription system PCR tube into the PCR instrument and set the reaction program as follows: a) incubate at 50°C for 15 minutes; b) inactivate at 85°C for 10 seconds. The mRNA in the system is reverse transcribed into cDNA.

[0136] 5. Fluorescence quantitative PCR

[0137] (1) Using the reagents in the SYBR Green fluorescent quantitative assay kit, prepare the reaction system in an eight-tube PCR tube containing: a) 2× SYBR Green PCR premix, 10 μL; b) forward and reverse primers, 1.5 μL each; c) 1 μg / μL cDNA solution, 1 μL; d) sterilized first-grade water, 6 μL. The final PCR volume is 20 μL. Briefly centrifuge for 30 seconds using a handheld centrifuge.

[0138] (2) Each sample requires three replicate wells for each molecule to be tested, and a blank control without adding cDNA is set up; in addition to the molecule to be tested, actin β (ACTB) is also required as a reference molecule.

[0139] (3) Place the eight-tube PCR tubes in a fluorescent quantitative PCR instrument and set the reaction program: a) 95°C for 2 minutes; b) 95°C for 5 seconds; c) 60°C for 10 seconds; d) 20°C for storage. Repeat steps b and c for 45 cycles.

[0140] (4) After the PCR process is completed, the software automatically generates the CT value for each sample.

[0141] 6. Quantitative Analysis of Results

[0142] (1) The CT values ​​of three replicates for each sample were averaged, and the difference between the CT value of the test molecule and the CT value of the reference molecule (ACTB) was calculated, i.e., ΔCT.

[0143] (2) Calculate the difference between the ΔCT value of the molecule to be tested in the cells after treatment with the raw material and the ΔCT value of the molecule to be tested in the untreated cells, i.e., ΔΔCT.

[0144] (3) Calculate the exponential value with 2 as the base and (-ΔΔCT) as the exponent, that is, 2 -△△CT , 2 parallel groups of each concentration -△△CT The average value is taken as the quantitative result of the molecule to be tested in each sample to achieve quantitative detection at the gene level.

[0145] The experimental results are shown in Table 6 below.

[0146] Table 6

[0147]

[0148]

[0149] As shown in Table 6, by treating cells with different concentrations of collagen and measuring gene expression, the concentration range of 0.2mg / mL to 0.8mg / mL ensured that the expression of all target genes was promoted. Among them, 0.5mg / mL showed the highest promotion rate.

[0150] Example 4

[0151] This example uses the recombinant collagen obtained in Example 1 to conduct a fibroblast proliferation experiment to verify the effects of different concentrations of recombinant collagen on fibroblast proliferation. The experimental steps include:

[0152] (1) Digest BJ fibroblasts that have entered the exponential culture phase with trypsin for about 20 seconds, add DMEM medium to terminate digestion, and resuspend for later use.

[0153] (2) Place a 2-well cell scratch insert in the center of each well of a six-well cell culture dish, and slowly drip the resuspended BJ cells into the well. After culturing for 24 hours until the cells are completely attached to the wall, remove the scratch insert and add 2 mL of DMEM culture medium to each well. Add 1% sterile water to the control wells. Add 1% of 0.2 mg / mL, 0.5 mg / mL, and 0.8 mg / mL recombinant collagen solutions to the experimental wells, respectively. Place in a cell culture incubator and culture for 24 hours. Repeat the experiment twice for each group.

[0154] (3) Observe and calculate the cell scratch recovery. The results are as follows Figures 2-9 The statistical data of scratch width (unit / micrometer) are shown in Table 7.

[0155] Table 7

[0156]

[0157] The recovery rate in Table 7 is calculated as follows: recovery rate = (0-hour mean - 24-hour mean) / 0-hour mean.

[0158] From the results in Table 7, it can be seen that recombinant collagen has a significant effect on promoting the proliferation of fibroblasts and can restore the scratches of fibroblasts more quickly.

[0159] Example 5

[0160] This example uses the recombinant collagen obtained in Example 1, as well as collagen 1, collagen 2, and collagen 3, to simultaneously test their effects on promoting the expression of a series of genes related to skin fibroblast proliferation.

[0161] The amino acid sequence of collagen 1 is shown in SEQ ID NO: 2; the amino acid sequence of collagen 2 is shown in SEQ ID NO: 3; and the amino acid sequence of collagen 3 is shown in SEQ ID NO: 4.

[0162] The experimental steps include:

[0163] 1. The recombinant collagen obtained in Example 1, as well as collagen 1, collagen 2, and collagen 3, were prepared into 0.5 mg / mL test solutions. The solutions were added to a culture medium of BJ cells that were growing adherently to approximately 70% of the bottom area of ​​the culture dish at a final concentration of 0.1% and cultured for 24 hours.

[0164] 2. Subsequent RNA extraction, reverse transcription, and qPCR experimental procedures were the same as those described in Example 3. The experimental results are shown in Table 8.

[0165] Table 8

[0166] Gene name Collagen 1 Collagen 2 Collagen 3 Recombinant collagen BMP2 1.0562734 1.000666701 1.066354872 1.3534789 CCL2 1.630 2.923312155 1.186 3.239 CCN1 1.658 2.097748796 1.294 2.149 CCND1 1.138 1.406657597 0.990 1.579 EDN1 1.103 1.483990484 1.590 1.610 EDN2 3.304 2.960170724 2.053 4.899 FGF1 1.194 1.1461919 1.046 1.629 FOXC2 1.060 1.048093457 1.182 1.305 FOXF1 1.244 1.275773295 1.002 1.531 HGF 1.031 0.98381206 1.107 1.319 KLF4 1.642 1.762915839 1.502 2.012 LIF 1.560 1.739070287 1.503 1.837 MYC 1.327 1.243265916 1.615 1.738 NGF 1.141 1.163790948 1.255 1.399 NOG 2.321 1.572934147 1.517 2.757 PDGFA 1.295 1.060638894 1.131 1.498 PDGFB 1.189 1.394875664 1.058 1.738 RUNX2 1.223 1.005002722 1.415 1.489 SERPINE1 1.063 0.997182978 1.226 1.398 SFRP1 1.607 1.098984148 1.328 1.769 SMAD3 1.301 1.238970938 1.232 1.448 SNAI1 1.220 1.281740576 1.083 1.397 STAT1 1.266 1.162384917 1.240 1.837 STAT3 1.255 1.36856676 1.686 1.740 TGFB1 1.316 1.118575767 1.435 1.578 TGFB2 1.004 1.157160274 1.146 1.236 VEGFA 1.436 1.437608877 1.291 1.659 Mean value 1.392 1.412225438 1.303 1.813

[0167] The qPCR results in the table show that the recombinant collagen of the present invention can upregulate 27 genes associated with skin fibroblast proliferation, while the other collagens, collagen 1, collagen 2, and collagen 3, cannot upregulate all 27 genes, and their upregulation effects are inferior to those of the recombinant collagen of the present invention. This indicates that the collagen of the present invention has a superior effect in promoting fibroblast proliferation, which is consistent with the conclusions in Example 3 above, indicating that the recombinant collagen of the present invention significantly enhances fibroblast proliferation.

[0168] Application Example 1

[0169] The recombinant collagen of the present invention can be used as an active ingredient in the preparation of skin conditioners and cosmetics, such as creams, lotions, gels, toners, essences, facial masks, eye creams, aerosol cleansing foams, sprays, shower gels, or facial cleansers.

[0170] Application 1. Preparation of facial cream: 0.1% recombinant collagen, 7% caprylic / capric triglyceride, 5% liquid paraffin, 4% glycerin, 3% propylene glycol, 3% ethylhexyl palmitate, 3% petrolatum, 2% ceteareth-21, 2% dimethicone, 2% cetearyl alcohol, 1.5% cetearyl ether-2, 1.5% glyceryl monostearate, 0.2% methylparaben, 0.2% carbomer, 0.2% triethanolamine, 0.1% ethylparaben, and the balance is water (65.2%).

[0171] Application 2, preparation of emulsion, formulated according to the following ratio: 0.1% recombinant collagen, 5% glycerin, 5% hydrogenated polyisobutene, 4% ethylhexyl palmitate, 3% propylene glycol, 3% caprylic / capric triglyceride, 2% polydimethylsiloxane, 1.5% ceteareth-21, 1.2% ceteareth-2, 0.5% tocopherol, 0.2% methylparaben, 0.1% triethanolamine, 0.1% ethylparaben, 0.1% carbomer, and the balance is water (74.2%).

[0172] Application 3, preparation of lotion, prepared according to the following ratio: 0.1% recombinant collagen, 5% glycerol, 3% propylene glycol, 0.2% diazolidinyl urea, and the balance being water (91.7%).

[0173] In summary, the present invention uses transcriptomics to detect the expression of a series of genes associated with skin fibroblast proliferation, demonstrating that recombinant collagen can promote the expression of genes associated with skin fibroblast proliferation. This collagen can be used in the development and application of a variety of cosmetic products, imparting the efficacy and effect of promoting skin fibroblast proliferation to these cosmetic products. Furthermore, this series of genes can also serve as a reference for future evaluation of the ability of other raw materials or cosmetics to promote skin fibroblast proliferation.

[0174] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. Application of recombinant collagen in the preparation of cosmetics, characterized in that: The amino acid sequence of the recombinant collagen is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The cosmetics can increase the expression of genes related to skin fibroblast proliferation, and the skin fibroblast proliferation-related genes include: any one or a combination of at least two of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA.

3. The use according to claim 1 or 2, characterized in that The concentration of the recombinant collagen in the cosmetic is 0.4-0.6 mg / mL.

4. The use according to claim 3, characterized in that The cosmetics include: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

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